RNA polymerase elongation during gene transcription may be hindered by the many proteins bound to DNA (roadblocks). Alternatively, displacement by RNA polymerase could inhibit the activity of a DNA-bound protein, and a transcription factor (TF), for example, might lose control of a promoter. The mechanism by which RNA polymerases elongate through roadblocks without compromising their regulatory function is poorly understood. Previous mechanistic, single-molecule studies have focused on RNA polymerase disrupting nucleosomes. However, nucleosomes, which are only found in eukaryotes, interact with DNA non-specifically, and are substrates for post-translational modifications that regulate chromatin remodelling and transcription of DNA. In contrast, many TFs from organisms spanning all kingdoms recognize specific sites on DNA to shape the genome and regulate transcription, and do not undergo chemical modifications regulated by complex pathways. Instead, they respond to environmental cues such as DNA supercoiling, concentration, and the presence of multiple operators to which they bind with different affinities and cooperatively. These tunable, cooperative interactions determine architectural DNA modifications such as DNA bending, wrapping and looping, the role of which has not been addressed in earlier studies on transcription roadblocks either in vivo or in vitro. The effect of three model TFs, the lac repressr (LacI), the l repressor and the 186 bacteriophage CI repressor, on transcriptional elongation by RNA polymerase (RNAP), will be compared and contrasted using magnetic tweezers (MT) and AFM imaging. These complementary techniques provide dynamic measurements of active complexes operating on single DNA molecules (MT), and detailed static images of nucleoprotein complexes adsorbed on a surface (AFM), and are the most direct macromolecular analyses for elucidating the mechanistic details by which RNAP elongates past a TF. The results of this investigation will help us (i) understand how transcriptional factors (TFs) generat complex responses in genomic contexts, and (ii) indicate new ways in which to manipulate genes and construct synthetic regulatory circuits for transcription. Therefore, the overall goal of this proposal is to understand how protein-protein cooperativity and protein-mediated long-range interactions, such as DNA looping may affect the strength of a roadblock, and if DNA tension and transcription-generated DNA supercoiling may facilitate RNAP elongation through these TFs. Aim 1 will focus on the effects of TF binding affinity, looping, DNA tension and handedness of DNA supercoiling on the strong LacI roadblock. Aim 2 will focus on the effects of TF binding affinity, oligomerization, looping, DNA tension and handedness of DNA supercoiling on the weak l CI roadblock. Aim 3 will focus on the effects of alternate wrapping or looping, DNA tension and handedness of DNA supercoiling on the 186 CI repressor.